pVAX-eNOS转染急性心肌梗死患者早期内皮祖细胞及对其活性的影响

摘要

目的 探讨利用pVAX1 介导一氧化氮合酶(eNOS)基因体外转染急性心肌梗死患者(AMI)外周血早期内皮祖细胞(EPCs)的可行性.方法 选择陕西省人民医院2017年12月～2018年6月因心脏病发病入院且经冠脉造影检查确诊为AMI 患者30例.Ficoll 密度梯度离心法分离患者空腹静脉外周血单个核细胞诱导培养至EPCs 并鉴定,纯化pVAX-eNOS 质粒.将EPCs 分为3组,eNOS 转染组:用阳离子聚合物JetPEITM 体外转染eNOS 基因至EPCs.转染后24 h,硝酸还原酶法检测EPCs 中一氧化氮(NO)的含量,ELISA和Western blot 检测EPCs 分泌eNOS及血管内皮生长因子(VEGF)的能力;采用Wst-1 法检测EPCs 增殖情况,用Transwell 小室检测各组EPCs 迁移及黏附功能;空质粒转染组:只转染pVAX 空质粒至EPCs 中;对照组:不转染EPCs.结果 成功转染pVAX-eNOS基因至AMI 患者早期EPCs,转染后24 h,与空质粒转染组及对照组比较,NO 含量明显升高(P ＜ 0.01),eNOS 表达量明显增加(P ＜ 0.01),分泌VEGF的能力明显增强(P ＜ 0.01);EPCs 增殖、迁移及黏附能力明显增加(P ＜ 0.01),空质粒转染组与对照组比较,差异无统计学意义(P ＞ 0.05).结论 阳离子聚合物JetPEITM 可成功介导pVAX-eNOS 有效转染AMI 患者早期EPCs,并有效增强EPCs 增殖、迁移、黏附及分泌功能,为AMI 患者进行自体EPCs 移植改善心肌缺血奠定了基础.%Objective To investigate the feasibility of transfection of early endothelial progenitor cells (EPCs) in peripheral blood of patients with acute myocardial infarction (AMI) in vitro using pvax1-mediated nitric oxide synthase (eNOS) gene. Methods From December 2017 to June 2018, 30 cases of AMI patients admitted to Shaanxi Provincial People's Hospital due to heart disease and diagnosed by coronary angiography were selected. The Ficoll density gradient centrifugation method was used to isolate mononuclear cells in the peripheral blood of patients with fasting veins and induce them to culture into EPCs for identification and purification of pvax-enos plasmids. EPCs were divided into three groups: eNOS transfection group, cationic polymer JetPEITM was used to transfect eNOS gene into EPCs in vitro. After transfection for 24 h, nitric oxide (NO) content in EPCs was detected by nitrate reductase method, and the ability of eNOS and vascular endothelial growth factor (VEGF) secretion by EPCs was detected by ELISA and Western blot. The proliferation of EPCs was detected by Wst-1 method, and the migration and adhesion of EPCs in each group were detected by Transwell chamber. Empty plasmid transfection group, only pVAX empty plasmid was transfected into EPCs. Control group, no transfection of EPCs. Results Pvax-enos gene was successfully transfected into early EPCs of AMI patients, and 24 h after transfection, the content of NO was significantly increased compared with the blank plasmid transfection group and the control group (P ＜ 0.01), The expression of eNOS was significantly increased (P ＜ 0.01), and the ability to secrete VEGF was significantly increased (P ＜ 0.01). The ability of proliferation, migration and adhesion of EPCs was significantly increased (P ＜ 0.01), and there was no significant difference between the empty plasmid transfection group and the control group (P ＞ 0.05). Conclusion Cationic polymer JetPEITM can successfully mediate the effective transfection of pvax-enos into early EPCs in AMI patients, and effectively enhance the proliferation, migration, adhesion and secretion of EPCs, which lays a foundation for the improvement of myocardial ischemia in AMI patients by autologous EPCs transplantation.